Phthalamic Acid Research Articles

Synthesis and rearrangements of the pentaamminecobalt(III) linkage isomers of some amidic acids

The unidentate pentaamminecobalt(III) linkage isomers of phthalamic and malonamic acids bonded through the deprotonated amide nitrogen or the carboxylate group have been synthesized and characterized. The carboxylate-bonded complexes were synthesized directly from their amidic acids; they, as well as carboxylate-bonded succinamic acid, solvolyzed very slowly in aqueous acid ( 1 M, t 1 2 days) and at pH 10 they decomposed very slowly; the amide group was not hydrolyzed while the amidic acid ligand was bonded to the metal. The malonamato- N complex was prepared by base hydrolysis at pH 7 of the corresponding cyanoacetate- N complex. Pentaammine (phthalamato- N)cobalt(III) was synthesized by base catalyzed hydration of the corresponding phthalimido- N complex; the latter was prepared directly from free ligand and pentaammine (dimethyl sulfoxide) cobalt(III). The rate law for the base catalyzed hydration (10 −7 M ≤ [OH −] ≤ 0.05 M) of the phthalimido- N complex is: k ohs = k OH [OH −] where K OH = 0.40 M −1 s 1 (25°C, I = 1.0 M (NaCl). In acid the amido- N linkage isomers are protonated on both the amide oxygen and on the carboxylate group. In water they undergo the usual solvolysis and amide- N to O rearrangement, but, concurrently, amide- N to carboxylate-bonded rearrangement. This first-formed amide- O complexes aquate relatively rapidly so that the observed products were the pentaammineaquacobalt(III) complex and the carboxylate-bonded complexes of malonamic acid (73%), phthalamic acid (36%) and succinamic acid (24%), respectively. In Me 2SO-d 6 amide- N to O and amide- N to carboxylate-bonded rearrangements were observed directly, with the ultimate products being pentaammine (dimethyl sulfoxide) cobalt(III) and the carboxylate-bonded complexes of malonamic acid (10%), phthalamic (33%) and succinamic acid (5%), respectively. The effects of a one-versus two-C length organic backbone, flexible (−CH 2CH 2−) versus rigid ( o-phenylene) geometry, and solvent (H 2O versus Me 2SO) in influencing the competitive rearrangements are considered.

Spontaneous cyclodehydration ofN-(o-aminophenyl) amic acids

The model reaction between phthalic anhydride and 1,2-diaminobenzene was studied under conditions analogous to those of the low-temperature polycondensation of aromatic dianhydrides with bis(o-diamines) in amide solvents followed by thermal cyclodehydration in condensed state to form ladder polyimidazopyrrolones (polypyrrones). The intermediate N-(o-aminophenyl)phthalamic acid was found to undergo spontaneous cyclodehydration to give N-(o-aminophenyl)phthalimide and 2-(o-carboxy-phenyl)benzimidazole. The reaction occurred at ambient temperature in the presence of water or alcohol without using a dehydration agent. The yield of imide-amine and/or carboxy-benzimidazole depended on the temperature of the condensation reaction. Temperatures below 0°C appeared to favor the formation of the carboxy-benzimidazole. Thermal cyclization of N-(o-aminophenyl)phthalamic acid passed through the same intermediates: imide-amine and carboxy-benzimidazole. The former converted to corresponding pyrrone above 200°C, while the secondary cyclization of the latter started above 250°C. Spontaneous cyclodehydration was also observed for polyamide acid-amine precursors obtained by low-temperature polycondensation of pyromellitic dianhydride with 3,3′-diaminobenzidine. The prepolymer solutions in DMF diluted with water at room temperature turned to a gel after 48–72 h. A spectroscopic study of the resulting polymers indicated the presence of significant amounts of imide cycles. © 1998 John Wiley & Sons, Inc. J Appl Polym Sci 67: 609–619, 1998

Spontaneous cyclodehydration of N-(o-aminophenyl) amic acids

The model reaction between phthalic anhydride and 1,2-diaminobenzene was studied under conditions analogous to those of the low-temperature polycondensation of aromatic dianhydrides with bis(o-diamines) in amide solvents followed by thermal cyclodehydration in condensed state to form ladder polyimidazopyrrolones (polypyrrones). The intermediate N-(o-aminophenyl)phthalamic acid was found to undergo spontaneous cyclodehydration to give N-(o-aminophenyl)phthalimide and 2-(o-carboxy-phenyl)benzimidazole. The reaction occurred at ambient temperature in the presence of water or alcohol without using a dehydration agent. The yield of imide-amine and/or carboxy-benzimidazole depended on the temperature of the condensation reaction. Temperatures below 0°C appeared to favor the formation of the carboxy-benzimidazole. Thermal cyclization of N-(o-aminophenyl)phthalamic acid passed through the same intermediates: imide-amine and carboxy-benzimidazole. The former converted to corresponding pyrrone above 200°C, while the secondary cyclization of the latter started above 250°C. Spontaneous cyclodehydration was also observed for polyamide acid-amine precursors obtained by low-temperature polycondensation of pyromellitic dianhydride with 3,3′-diaminobenzidine. The prepolymer solutions in DMF diluted with water at room temperature turned to a gel after 48–72 h. A spectroscopic study of the resulting polymers indicated the presence of significant amounts of imide cycles. © 1998 John Wiley & Sons, Inc. J Appl Polym Sci 67: 609–619, 1998

Studying early nodulin gene ENOD40 expression and induction by nodulation factor and cytokinin in transgenic alfalfa.

ENOD40, an early nodulin gene, is expressed following inoculation with Rhizobium meliloti or by adding R. meliloti-produced nodulation (Nod) factors or the plant hormone cytokinin to uninoculated roots. We isolated two MsENOD40 clones, designated MsENOD40-1 and MsENOD40-2, with distinct promoters from an alfalfa (Medicago sativa cv Chief) genomic library. The promoters were fused to the reporter gene uidA (gus), and the constructs were introduced into alfalfa. We observed that the MsENOD40-1 construct was expressed almost exclusively under symbiotic conditions. The MsENOD40-2 construct was transcribed under both symbiotic and nonsymbiotic conditions and in nonnodular and nodular tissues. Both MsENOD40 promoter-gus constructs were similarly expressed as nodules developed, and both were expressed in roots treated with 6-benzylaminopurine or purified Nod factor. However, no blue color was detected in nodule-like structures induced by the auxin transport inhibitor N-1-(naphthyl)phthalamic acid on roots of plants containing the MsENOD40-1 promoter construct, whereas pseudonodules from plants containing the MsENOD40-2 promoter construct stained blue. A 616-bp region at the distal 5' end of the promoter is important for proper spatial expression of MsENOD40 in nodules and also for Nod-factor and cytokinin-induced expression.

Thermal decomposition of immobilized compounds for the generation of gaseous standard mixtures containing ammonia and amines

The thermal decomposition of phthalamic acid derivatives chemically bonded to the surface of silica gel was examined and utilized for the generation of single-component gaseous standard mixtures of ammonia, methylamine, diethylamine and triethylamine. The conditions of the thermal decomposition (temperature, time, diluent gas flow rate) were optimized to ensure complete liberation and rapid elution of the compounds from the bed of modified silica gel. The total amounts of these four compounds that can be released from unit mass of the modified silica gel are in the range of several mg.

A new kinetic model for the acid-catalysed reactions of N-(2-aminophenyl)phthalamic acid in aqueous media

The acid-catalysed breakdown of N-(2-aminophenyl)phthalamic acid has been studied in dilute aqueous acids in the pH range 0–6. The dominant reaction is the formation of N-(2-aminophenyl)phthalimide (between ∼80 and ∼100% yields in the pH range studied) and its subsequent rearrangement to 2-(2-carboxyphenyl)benzimidazole, occurring as consecutive pseudo-first-order processes. Anomalously, only a minor hydrolysis reaction is observed.A kinetic model for these processes has been constructed and rate constants and activation parameters evaluated. Mechanisms involving pre-equilibria to form the kinetically significant species have been proposed for the consecutive processes. The approach has been adapted to account for the observed kinetics of acid catalysed formation of benzimidazoles from o-aminoanilides.

Suggested Improvement in the Ing-Manske Procedure and Gabriel Synthesis of Primary Amines: Kinetic Study on Alkaline Hydrolysis of N-Phthaloylglycine and Acid Hydrolysis of N-(o-Carboxybenzoyl)glycine in Aqueous Organic Solvents.

A slight modification of the Gabriel synthesis of primary amines is suggested on the basis of the observed and reported values of rate constants for the alkaline and acid hydrolyses of phthalimide, phthalamic acid, benzamide, and their N-substituted derivatives. The suggested procedure requires shorter reactions time and milder acid-base reaction conditions compared with the conventional acid-base hydrolysis in the Gabriel synthesis. A slight modification in the Ing-Manske procedure is also suggested. Pseudo-first-order rate constants, k(obs), for hydrolysis of N-phthaloylglycine, NPG, decrease from 24.1 x 10(-3) to 7.72 x 10(-3) and 6.12 x 10(-3) s(-1) with increasing acetonitrile and 1,4-dioxan contents, respectively, from 2 to 50% v/v (all the percentages given in the paper are vol %), while increasing the organic cosolvents content from 50 to 80% increases k(obs) from 7.72 x 10(-3) to 19.7 x 10(-3) s(-1) for acetonitrile and from 6.12 x 10(-3) to 52.8 x 10(-3) s(-1) for 1,4-dioxan, in aqueous organic solvents containing 0.004 M NaOH at 35 degrees C. The rate constants for NPG hydrolysis decrease from 2.11 x 10(-2) to 1.19 x 10(-4) s(-1) with increasing MeOH content from 2 to 84%, in aqueous organic solvents containing 2% MeCN and 0.004 M NaOH at 35 degrees C.

Maturation‐related loss in rooting competence by loblolly pine stem cuttings: The role of auxin transport, metabolism and tissue sensitivity

A comparison of rooting ability of stem cuttings made from hypocotyls and epicotyls from 50‐day‐old seedlings of loblolly pine (Pinus taeda L.) reveals a dramatic decline by epicotyl cuttings, which do not root at all in 20–30 days in the presence or absence of auxin. In contrast, almost all the hypocotyls root during this time, but only in the presence of exogenously applied auxin. The failure of epicotyls to root does not appear to be due to differences in [14C]‐labeled auxin uptake, transport, metabolism, or tissue distribution in the two types of cuttings. At the cellular level, initial responses to auxin, such as differentiation of the cambium into parenchyma, occur in both types of cuttings, but localized rapid cell division and root meristem organization are not observed in epicotyls. Autoradiography revealed that radioactivity from a‐naphthalene acetic acid is bound in the cortex but not concentrated at sites of root meristem organization prior to the organization of the meristem in hypocotys. During the development of the epicotyl. cellular competence to form roots appears to be lost. Although this loss in competence is not associated with a concurrent loss in ability to transport auxin polarly, the latter process appears to play a key role in rooting other than to move auxin to the site of root formation. The phytotropin N‐(1‐naphthyl)phthalamic acid inhibits rooting if applied during the first 3 days after the cutting is made, but does not affect auxin concentration or metabolism at the rooting site.

The Auxin Transport Inhibitor N-(1-Naphthyl)phthalamic Acid Elicits Pseudonodules on Nonnodulating Mutants of White Sweetclover.

The collection of symbiotic (sym) mutants of white sweetclover (Melilotus alba Desr.) provides a developmental sequence of mutants blocked early in infection or nodule organogenesis. Mutant phenotypes include non-nodulating mutants that exhibit root-hair deformations in response to Rhizobium meliloti, mutants that form ineffective nodules lacking infection threads, and mutants that form infection threads and ineffective nodules. Mutant alleles from both the sym-1 and the sym-3 loci exhibited a non-nodulating phenotype in response to R. meliloti, although one allele in the sym-1 locus formed ineffective nodules at a low frequency. Spot-inoculation experiments on a non-nodulating allele in the sym-3 locus indicated that this mutant lacked cortical cell divisions following inoculation with R. meliloti. The auxin transport inhibitor N-(1-naphthyl)phthalamic acid elicited development of pseudonodules at a high frequency on all of the sweetclover sym mutants, including the non-nodulating mutants, in which the early nodulin ENOD2 was expressed. This suggests that N-(1-naphthyl)phthalamic acid activates cortical cell divisions by circumventing a secondary signal transduction event that is lacking in the non-nodulating sweetclover mutants. The sym-3 locus and possibly the sym-1 locus appear to be essential to early host plant responses essential to nodule organogenesis.

Quinine as a chiral additive in nonaqueous capillary zone electrophoresis

Quinine was used as a chiral additive in nonaqueous capillary zone electrophoresis. The separation buffer consisted of ammonium acetate and quinine in methanol. Quinine was able to enantioresolve a number of N-3,5-dinitrobenzoylated amino acids as well as (±)-1,1′-binaphthyl-2,2′diyl hydrogen phosphate and N-[1-(1-naphthyl)ethyl]phthalamic acid. Enantioresolution deteriorated as the concentration of ammonium acetate was increased but improved with the addition of acetic acid. Chiral recognition seems to arise through an ion-pairing mechanism. © 1996 John Wiley & Sons, Inc.

Kinetic Study on the Hydrolysis of 1-N-Naphthylphthalamic Acid (Naptalam)

The hydrolysis rate of 1-N-naphthylphthalamic acid (Nap) was measured as a function of pH in the range of 0.9-5 and proceeds via undissociated neighboring carboxyl group participation as was proposed for other phthalamic acid derivatives. One of the main hydrolysis products is naphthylamine, which is a known carcinogenic agent. Nap is as reactive as phthalamic acid but is less reactive than phthalanilic acid. MMX calculations on phthalamic acid derivatives showed that phthalanilic acid has the closest distance for a most efficient intramolecular catalysis. Additives such as cyclodextrin or metal ion do not affect the kinetics of the reaction.

In vitroでのブドウ'デラウェア'子房の生長におけるジベレリンとオーキシンの共働作用

The correlative effect of gibberellin A3 (GA3) and indole-3-acetic acid (IAA) on the development of 'Delaware' grape ovary was examined in vitro. GA3 induced a significant increase of the ovary fresh weight, proportionate to its concentration; IAA and tryptophan (Trp), a precursor of IAA, had little effect on increasing in fresh weight of the ovary. GA3 + IAA increased the ovary fresh weight more than GA3 or IAA alone. The fresh weights of the GA3 or IAA treated ovaries increased 1.08- to 1.56-fold over the control, whereas those treated with the combination of GA3 and IAA increased 1.85- to 2-fold over control after a 9-10 week-culture. The effect of GA3+ Trp on increasing in fresh weight of the ovary was about the same as that of GA3+IAA. These results indicate that GA3 promotes the ovary development more than IAA and that IAA and Trp enlarge the promotive effect of GA3.The ovary development in response to GA3 and GA3 + Trp was partly inhibited by anti-auxins such as N-(1-naphthyl) phthalamic acid (NPA), 2, 3, 5-triiodobenzoic acid (TIBA). Besides inducing an increase of the ovary fresh weight, GA3 increased the content of IAA-like substances in the ovary. From these results, it appears that the effect of GA3 on the ovary development is ascribed not only to its original action, but also to the synergistic action with IAA synthesized de novo.

Multicomponent determination of the pesticide naptalam and its metabolites in river water, by applying partial least squares calibration to the derivative spectrophotometric signals

A multivariate calibration method, partial least squares (types PLS-1 and PLS-2), was applied to the simultaneous determination of naptalam (N-(1-naphthyl) phthalamic acid) and its metabolites N-(1-naphthyl) phthalimide and 1-naphthylamine in mixtures by UV-visible absorption spectrophotometry. The absorption and first-derivative absorption spectra of mixtures were used to perform the optimization of the calibration matrices by the PLS method. Two different experimental designs for the three-component mixtures are assayed and the results are discussed. The proposed method with the derivative spectra was applied to the determination of these analytes in river water at the ppb level.

A Gene That Encodes a Proline-Rich Nodulin with Limited Homology to PsENOD12 Is Expressed in the Invasion Zone of Rhizobium meliloti-Induced Alfalfa Root Nodules

To define the early stages of the interaction between Rhizobium and host legumes, we have cloned and characterized three early nodulin-encoding sequences from an alfalfa (Medicago sativa L.) cDNA library by probing with a fragment of a cDNA clone for PsENOD12, an infection-related nodulin from pea (Pisum sativum L.). Although the coding regions of the three clones are 95 to 98% homologous to each other, they are only 43% homologous to the pea clone. However, the putative signal peptide encoded by the alfalfa cDNA clones is 100% homologous to the PsENDO12 signal peptide. The spatial and temporal expression patterns of PsENOD12 and the alfalfa clones were compared. In situ hybridization experiments detected RNA transcripts in the invasion zone of mature nitrogen-fixing nodules, the same site where PsENOD12 mRNAs are found. Transcripts were also found by in situ hybridization in cells of Rhizobium meliloti exoH mutant-induced nodules penetrated by infection threads, but northern analysis did not detect transcripts in inf- (infection thread minus) nodules elicited by R. meliloti exoB nodules or in pseudonodules elicited by treatment with the auxin transport inhibitor N-1-(naphthyl)phthalamic acid. In addition, the alfalfa gene represented by these cDNA clones exhibited a temporal expression pattern that differed from that of PsENOD12, which is transiently expressed. These data, plus information derived from Southern blot analysis, indicate that we have isolated cDNA clones for a novel early nodulin, which we have designated MsENOD10 (Medicago sativa Early Nodulin 10).

The PsENOD12 Gene Is Expressed at Two Different Sites in Afghanistan Pea Pseudonodules Induced by Auxin Transport Inhibitors

A number of early nodulin genes are expressed in specific cell types as pea (Pisum sativum) root nodules develop. The Pisum sativum early nodulin PsENOD2 is detected only in the uninfected cells of the nodule parenchyma, whereas PsENOD12 is expressed at two spatially removed sites: in root hairs and adjacent cortical cells, both of which can be invaded by Rhizobium entering through infection threads, and in derivatives of newly divided root inner cortical cells that establish the nodule primordium. We tested whether Rhizobium infection is required for triggering PsENOD12 gene expression by inducing nodule-like structures on Afghanistan pea roots with the auxin transport inhibitor N-(1-naphthyl)phthalamic acid (NPA). These nodule-like structures lack infection threads but resemble Rhizobium-induced nodules in other aspects. For one, both PsENOD2 and PsENOD12 transcripts were detected in these structures. PsENOD2 mRNA was localized by in situ hybridization to a zone equivalent to the nodule parenchyma of Rhizobium-induced nodules, whereas PsENOD12 transcripts were detected in a group of cells comparable to the nodule primordium of developing nodules. In addition, PsENOD12 mRNA was detected in uninfected root hairs 48 h after NPA treatment. These results indicate that infection is not a trigger for PsENOD12 gene expression in Afghanistan pea and rather suggest that the expression of the PsENOD2 and PsENOD12 genes is correlated with the differentiation of specific cell types in the developing nodule.

Rationale for the chemical development of angiotensin II receptor antagonists.

The renin-angiotensin system (RAS) has been demonstrated to be a key element in blood pressure regulation and fluid volume homeostasis. Since angiotensin II (AII) is the effector molecule of the RAS, the most direct approach to block this system is to antagonize AII at the level of its receptor. Therefore, at Du Pont Merck the working hypothesis has been that the identification of metabolically stable and orally effective AII-receptor antagonists would constitute a new and superior class of agents useful in treating hypertension and congestive heart failure. Our program began with a detailed pharmacologic evaluation of some simple N-benzylimidazoles, originally described by Takeda Chemical Industries in Osaka, Japan. They were found to be a series of weak but selective AII-receptor antagonists with a competitive mode of action. We embarked on a program aimed to design and synthesize more potent and orally effective nonpeptide antagonists, while attempting to preserve their selective affinity for the AII receptor. The first major breakthrough in our efforts to increase the potency of these compounds came with the development of a series of N-benzylimidazole phthalamic acid derivatives. Although effective at lowering blood pressure when administered intravenously, the phthalamic acids were devoid of oral activity. The first orally active AII antagonists came with the discovery of the biphenyl carboxylic acids. Although these compounds are absorbed after oral dosing, their bioavailability was less than desired. In the hope of improving the oral absorption of these biphenyls, we investigated a variety of acidic groups as bioisosteric replacements for the carboxylic acid. The key to the discovery of nonpeptide AII-receptor antagonists with improved oral activity and duration of action resulted from replacing the carboxylic acid group with the isosteric but more lipophilic tetrazole ring. Hence, our efforts culminated in the discovery of losartan (2-n-butyl-4-chloro-5-hydroxymethyl-1-[(2'-(1H-tetrazol-5-yl) biphenyl-4-yl)methyl]imidazole, potassium salt), a highly potent angiotensin type 1 (AT1) selective receptor antagonist with a long duration of action. Losartan is currently undergoing clinical investigation for the treatment of hypertension. The history, including the rationale for the design of the compounds, and ensuing structure-activity relationships of losartan and related analogs will be described. Many of the newer compounds exceed the potency of losartan, and the best compounds in the series rival the affinity of the endogenous ligand, AII, for its receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Requirement of the Auxin Polar Transport System in Early Stages of Arabidopsis Floral Bud Formation.

The pin-formed mutant pin 1-1, one of the Arabidopsis flower mutants, has several structural abnormalities in inflorescence axes, flowers, and leaves. In some cases, pin1-1 forms a flower with abnormal structure (wide petals, no stamens, pistil-like structure with no ovules in the ovary) at the top of inflorescence axes. In other cases, no floral buds are formed on the axes. An independently isolated allelic mutant (pin1-2) shows similar phenotypes. These mutant phenotypes are exactly the same in wild-type plants cultured in the presence of chemical compounds known as auxin polar transport inhibitors: 9-hydroxyfluorene-9-carboxylic acid or N-(1-naphthyl)phthalamic acid. We tested the polar transport activity of indole-3-acetic acid and the endogenous amount of free indole-3-acetic acid in the tissue of inflorescence axes of the pin1 mutants and wild type. The polar transport activity in the pin 1-1 mutant and in the pin1-2 mutant was decreased to 14% and 7% of wild type, respectively. These observations strongly suggest that the normal level of polar transport activity in the inflorescence axes is required in early developmental stages of floral bud formation in Arabidopsis and that the primary function of the pin1 gene is auxin polar transport in the inflorescence axis.

Inhibition of auxin transport by isoquinolinedione herbicides

2-(p-carbethoxyphenyl)-1,3(2H,4H)-isoquinolinedione (CEPIQ), an experimental herbicide, caused effects on geotropism, which are often indicative of an effect on auxin transport, in a whole plant herbicidal screen. However, it showed little or no activity in an in vitro binding assay in corn coleoptiles for the auxin-transport inhibitor,N-1-naphthylphthalamic acid (NPA). Other active isoquinolinedione analogues of this compound did, however, exhibit significant in vitro activity. Direct measurements of auxin transport in corn coleoptiles were undertaken in an attempt to resolve the apparent discrepancy between herbicidal and binding activities. In all cases examined, compounds that were highly active on whole plants were good inhibitors of auxin transport, and compounds that were weak as herbicides showed little or no effect on auxin transport. Therefore, it is concluded that the mode of action of these isoquinolinedione herbicides is the inhibition of auxin transport. Ring-opened analogues of several isoquinolinediones were synthesized and assayed in both the transport and binding assays, in order to test whether compounds in this class express their herbicidal activity by undergoing ring-opening in vivo, yielding products that are more straightforward analogues of NPA with free carboxyl groups. The homophthalamic acids had little or no activity in both assays. On the other hand, thep-ethyl- andp-ethoxy-phenyl phthalamic acids showed auxin transport inhibition comparable to the parent isoquinolinediones, but with markedly increased binding activity. These results support the possible role of ring-opening in the generation of biological activity. However, thep-carbethoxyphenyl phthalamic acid, analogous to CEPIQ, was very weak in both assays. Thus, ring-opening in vivo cannot alone account for the biological activity of this class of compounds.

Differential regulation of a fruit‐specific 62 kDa protein in developing parthenocarpic (pat‐2/pat‐2) and seeded tomato fruits

The denatured protein profiles of developing tomato (Lycopersicon esculentum Mill.) fruits, from the anthesis stage up to fruits at 30% of their final diameter, were examined in a pai‐2l pat‐2 parthenocarpic line and in its near isogenic non‐partheno‐carpic line. At anthesis no differences were detected between the protein patterns of ovaries developed on parthenocarpic and non‐parthenocarpic plants. In subsequent stages the seeded and seedless fruits differed in the pattern of manifestation of several abundant proteins, none of which seem to be included in seeds The most prominent difference was found in an insoluble protein of 62 kDa; in developing seeded fruits of either the parthenocarpic or the non‐parthenocarpic line, its rate of decline was much faster than in seedless fruits. In seeded fruits larger than 4‐6 mm in diameter it was scarcely detected, whereas in parthenocarpic seedless 8–10 mm fruits it was still abundant. This protein is fruit specific; it is also enhanced in chemically (n‐n‐tolyl phthalamic acid) – induced parthenocarpic fruits of the non‐parthenocarpic line. The prolonged manifestation in the parthenocarpic fruits results from de novo synthesis of this protein. There are indications that it is not a stress‐related protein. This is the first demonstration of an association between the pattern of modulation of a protein and the phenotypic expression of genetically controlled parthenocarpy.

Nodulin Gene Expression and ENOD2 Localization in Effective, Nitrogen-Fixing and Ineffective, Bacteria-Free Nodules of Alfalfa.

Alfalfa plants form bacteria-free nodules in response to a number of agents, including Rhizobium meliloti exo mutants, Agrobacterium tumefaciens transconjugants carrying cloned R. meliloti nodulation genes, and compounds that function as auxin transport inhibitors, N-( 1-naphthyl)phthalamic acid or 2,3,5-triiodobenzoic acid. These bacteria-free nodules contain transcripts for the nodulins Nms30 and MsENOD2; transcripts for late nodulins like leghemoglobin are not detected. In situ hybridization studies demonstrated that ENOD2 transcripts were localized in parenchyma cells at the base and along the periphery of nitrogen-fixing alfalfa root nodules. The ENOD2 gene was also expressed in a tissue-specific manner in nodules elicited by N-( 1-naphthyl)phthalamic acid and 2,3,5-triiodobenzoic acid. In bacteria-free nodules induced by R. meliloti exo mutants and A. tumefaciens transconjugants carrying either one or both R. meliloti symbiotic plasmids, ENOD2 transcripts were also detected but were usually localized to parenchyma cells at the base instead of along the periphery of the nodule. On the basis of the pattern of ENOD2 gene expression, we conclude that the developmental pathway of bacteria-free nodules, whether bacterially or chemically induced, is the same as that of nitrogen-fixing nodules, and, furthermore, that the auxin transport inhibitors in their action mimic some factor(s) that trigger nodule development.